A fundamental problem faced by all organisms is the maintenance of genomic integrity. DNA damage intergeres with essential cellular processes, and unrepaired or misrepaired DNA can be mutagenic and carcinogenic. Defects in DNA repair associated with human cancer have been found in the cancer-prone diseases xeroderma pigmentation (XP) and nonpolyposis colon cancer. DNA repair proteins encoded by the human genes have yeast homologs which serve analogous functions. Therefore, yeast provides an excellent model system for studying eukaryotic DNA repair and its relationship to the disease state in humans. The overall goal of this proposed study is to elucidate the mechanism of transcription coupled repair in eukaryotes. 1) We will study the effects of altered expression of Rad26 in yeast. Mutations of the RAD26 gene (or ERCC6, its human homolog) results in cells that lack transcription-coupled repair. We will express RAD26 from a regulatable promoter in a rad26 mutant and explore its effect upon transcription-coupled repair and overall repair, under varying expression levels. We will also investigate the expression of a reporter gene Green Fluorescent Protein (GFP), in real time, under the same growth conditions. 2) We will study the effects of transcription rate upon transcription-coupled DNA repair in rad26 mutants. Unlike previous studies with inducible genes, transcription will be continuously increased from very low to very high rates of expression. 3) We will genetically identify factors that interact with Rad26 in vivo using selection for extragenic suppressors and a synthetic lethal screen. The two hybrid system will be used to identify proteins expressed from a yeast genomic library which interact with Rad26 protein. 4) We will investigate the role of chromatin structure in transcription- coupled DNA repair in rad26 mutants and identify domains of Rad26 that direct repair to transcriptionally active chromatin. Our in vivo approach eliminate a major hurdle to present biochemical approaches in that we are investigating the removal of DNA damage from bona fide chromatin substrates.